CN102899416B - Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof - Google Patents
Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof Download PDFInfo
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Abstract
The invention discloses cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and a rapid detection method thereof, which are specifically used for the specific detection of cucumber phytophthora. The rapid detection method is characterized in that a cucumber phytophthora LAMP primer is mainly adopted and designed, detailed information can be seen in SEQ NO.1-SEQ NO.4. Green fluorescent light or an LAMP characterized ladder-shaped pattern can be observed through the LAMP and the color developing by adding an SYBR green I color agent for developing color or the agarose gel electrophoresis detection. The LAMP primer and the rapid detection method, which are disclosed by the invention, can be used for quickly, sensitively and accurately detecting the cucumber phytophthora in plants and soil which are affected by the cucumber phytophthora in production practice and can be simultaneously used for the early diagnosis of diseases in filed and the monitoring and the identification of germs, and reliable technical and theoretical basis is provided for preventing the disease caused by the cucumber phytophthora.
Description
Technical field
The present invention relates to a kind of cucumber phytophthora LAMP primer and method for quick thereof, being exclusively used in cucumber phytophthora highly sensitive rapid molecular detects, can be used for the early diagnosis of field Cucumber Blight and the monitoring of germ and evaluation simultaneously, belong to corps diseases detection, evaluation and Prevention Technique field.
Background technology
Cucumber epidemic disease mould (
phytophthora meloniskatsura) by Katsura nineteen sixty-eight first from the cucumber of falling ill separation obtain, and be accredited as novel species, the state such as China, Japan, Egypt, Turkey, Korea S, India also reports this cause of disease in succession subsequently.By this microbial Cucumber Blight, be that one of the most serious disease occurs in Chinese cucumber producing region, stem, leaf and the fruit of the plant of mainly causing harm, all can occur from seedling stage to the strain phase.This germ survives among soil, short incubation period, and velocity of propagation is fast, and invasiveness is strong, destructive large to plant.In the applicable situation of humiture, at the utmost point, in the short period of time, can cause disease to be very popular, because epidemic disease causes harm the cucumber yield that loses up to 80%.Cucumber epidemic disease is mould can infect the crops such as cucumber, summer squash, hami melon, wax gourd.Morbidity plant is carried out disease screening fast and accurately, to the timely detection of pathogenic bacteria in anosis vegetable material and plant growth environment and monitoring, is to control that Plant diseases occurs, the popular and important foundation of causing disaster.Therefore set up cucumber phytophthora rapid detection system, in early days disease plant and soil are carried out to phytophthora and quick and precisely detect, to prediction disease a situation arises, take in time effectively preventing measure control pathogenic bacteria propagation and popular, reduce financial loss and all there is important theoretical and practical significance.
At present the detection of cucumber phytophthora is still continued to use to traditional cultivation and authentication method mostly, traditional detection of pathogens technology is to obtain, on the basis of pathogen, judging the kind of pathogen by morphological observation and Koch's Postulates in separation.Whole process usually needs to expend a large amount of labor forces and time, generally needs within several days, just can complete.And require operator to possess professional pathogenicbacteria separation, Morphological Identification knowledge and rich experience.Therefore, take morphological specificity as basic conventional disease screening technology, due to its length consuming time, efficiency is low, is difficult to meet the actual needs to Cucumber Blight diagnosis, because it is easy to miss the best period of disease control.Therefore, set up a set of quick, sensitive, accurately cucumber phytophthora to detect diagnostic techniques not only very necessary, and very urgent.
Round pcr provides quick, sensitive, advantage accurately for pathogenic diagnosis, yet PCR specific detection technology still needs professional instrument and the molecular biology reagent that PCR instrument, electrophoresis and gel imaging system etc. are expensive at present, and need molecular biology Specialty Experiment personnel operation, limited applying of PCR detection method.Circulation constant temperature amplification technique (Ioop-mediated isothermal amplification is called for short LAMP) is a kind of New Cycle constant temperature nucleic acid amplification technology of being developed by people such as the Japanese Rong Yan Notomi of Co., Ltd. for 2000.LAMP reaction is designed 4 primers for 6 sites of target gene, utilize a kind of chain type substitute activity archaeal dna polymerase (
bstdNA polymerase), under constant temperature, (60 65 ℃ of –) insulation 30 – is 90 minutes, can complete amplified reaction.High efficiency and isothermal rapid amplifying due to LAMP reaction can increase 10 in 90 minutes
9– 10
10times product.The detection of LAMP amplified production generally adopts the methods such as fluorescence dye visual observations, agarose gel electrophoresis and turbidity observation.Because LAMP reaction is simple, quick, efficient, economic dispatch feature, thereby there is application prospect very widely.LAMP detects and is mainly used in people and animals' pathogen, food safety and sanitary detection at present, and in phytopathogen detects, report is few, and the detection of cucumber phytophthora is not reported both at home and abroad.
Summary of the invention
The object of the invention is, detection method poor specificity long for the required cycle of biological detection method of cucumber phytophthora in prior art, the problem that sensitivity is low, provides a kind of LAMP of cucumber phytophthora to detect the method for quick of primer and reliable results, easy handling, high specificity, highly sensitive cucumber phytophthora.
Realizing object of the present invention comprises the following steps:
The design of 1.LAMP primer
It is mould that we download cucumber epidemic disease from GenBank
ypt(accession number: EF649778) gene order, mould according to cucumber epidemic disease
yptgene order, adopts a kind of LAMP of PrimerExplorer V4 software design to detect primer, comprises 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), and primer sequence is respectively:
F3：5’-AAATTCGCACGATCGAGCT-3’
B3：5’-CCGTGACGTCGTACACCA-3’
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’
2. the foundation of cucumber phytophthora rapid detection system
LAMP detects: each 0.2-0.25 μ M of F3 and B3 in 25 μ l reaction systems, each 1.5-1.7 μ M of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 10-50ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 60-65 ℃ of incubation 30-90 min, 80 ℃ of insulation 5-10min.
More preferably, each 0.2 μ M of F3 and B3 in 25 μ l reaction systems, each 1.6 μ M of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
Described detection method is fluorescence dye visual observations method and agarose gel electrophoresis method.Described fluorescence dye visual observations method: add 1 μ l developer in the final amplified production of LAMP reaction, described developer is SYBR green I, colour developing result is observed green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Described agarose gel electrophoresis method: get 2 μ l pcr amplification products and detect with 2% agarose gel electrophoresis, be judged as the positive if there is the distinctive trapezoid belt of LAMP, do not occur that amplified band is judged as feminine gender.
The highly sensitive rapid detection of the plant that the method can be used for carrying disease germs and soil.Set up that cucumber phytophthora is quick, easy, high specificity, highly sensitive Monitoring techniques system, for cucumber phytophthora, cause the early monitoring before the aobvious disease of disease, determine that disease control best period tool is of great significance.
Guardian technique of the present invention is primer sequence and the amplification method thereof of the efficient specific amplified of cucumber phytophthora.In order to verify the special primer sequence of cucumber phytophthora, the present invention be take 36 strain cucumber phytophthoras of the provinces such as China Fujian, Guangdong, Jiangsu and 22 kinds of different fungies and 11 kinds of phytophthoras and pythium oomycetes as for examination material, adopts CTAB method to extract the DNA of cucumber phytophthora in tissue.Concrete grammar is as follows: get hypha powder after 50mg lyophilize in 1.5ml centrifuge tube, (formula of extracting solution is: 2% CTAB to add 900 μ l 2% CTAB (cetyl trimethylammonium bromide) extracting solutions, 100 m mol/L Tris-HCl(Tri(Hydroxymethyl) Amino Methane Hydrochlorides), PH 8.0, 20mmol/L EDTA(disodium ethylene diamine tetraacetate), pH8.0, 1.4 mol/L NaCl) and 90 μ l 10% SDS(Sodium dodecylbenzene sulfonatees) after mix, in 55～60 ℃ of water-bath 1.5 h, every 10 min vibrations mix once, after water-bath 1.5h centrifugal (12, 000rpm) 15min, getting supernatant liquor adds and the isopyknic phenol/chloroform/primary isoamyl alcohol (phenol of supernatant liquor, the volume ratio of chloroform and primary isoamyl alcohol is 25:24:1), centrifugal (12, 000rpm) 5 min, get supernatant liquor (water), add and the isopyknic chloroform extracting of supernatant liquor once (12, 000rpm) centrifugal 5min, suct (350 μ l) clearly, add the 3mol/L NaAC solution of 0.1 volume (35 μ l) and the ice dehydrated alcohol of 2 volumes (700 μ l), at-20 ℃, precipitate after 30min 12, centrifugal 5 min of 000rpm, remove lightly supernatant liquor, add 700 μ l ice 70% ethanol to wash (slightly centrifugal, incline and fall supernatant), on Bechtop, naturally dry after alcohol-free taste with 1 * TE(10mmol/L Tris-HCl, 0.1mmol/L EDTA, pH8.0) solution dissolves, obtain DNA solution, by UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by.
Through the specificity of strains tested and 36 strain cucumber phytophthoras is carried out to LAMP checking.
LAMP reaction system 25 μ l: comprise each 0.2 μ M of F3 and B3, each 1.6 μ M of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water.
Except the 36 strain cucumber phytophthoras colour developings results from provinces such as China Fujian, Guangdong, Jiangsu can be observed green fluorescence or agarose gel electrophoresis, detect and occur the distinctive trapezoid belt of LAMP, it is orange having detected 22 kinds of fungal bacterial strains and 11 kinds of other oomycetes colour developing results or amplified band does not appear in agarose gel electrophoresis.This illustrates that this primer can be used to fast and reliable detection and the evaluation of cucumber phytophthora in incidence tissue and soil in production practice.
1. when when there is cucumber phytophthora for cucumber tissue, adopt the quick cracking process of NaOH to extract the DNA of cucumber phytophthora, detailed process is as follows: (1) is cleaned the sick leaf of cucumber or sick stem, dry, clip site of pathological change; (2) by the sick leaf of 1mg, add 10 μ l(0.5mol/L NaOH, 0.5%PVP) metering, by organizing, be fully milled to paste, centrifugal 5min in 12,000g whizzer; (3) get the Tris-HCl(pH8.0 of supernatant 20 μ l and isopyknic 0.1 mol/L) mix; (4) 10 times, 100 times, 1000 times liquid of dilution, get respectively 1 μ l stoste, 10 times, 100 times, 1000 times liquid increase as pcr template.
2. when when there is cucumber phytophthora for cucumber soil, adopt soil DNA extraction method to extract the DNA of banana blight bacteria in soil.Concrete grammar is as follows: after getting the freezing 24-48 of the draining h of the soil sieving, add a small amount of quartz sand, pour liquid nitrogen into and fully grind, the soil fine powder after grinding is divided and is filled in 1.5 ml centrifuge tubes, every pipe adds 500 μ l 0.4% skim-milk solution, and vortex mixes.Centrifugal 15 min of 12000 rpm.Get supernatant and add equal-volume Proteinase K damping fluid, adding final concentration is 10 μ g/ml Proteinase Ks, 55 ℃ of water-bath 1-3 h.After water-bath finishes, add 7.5 M NH of 1/2 volume
4aC solution, turns upside down and mixes.Centrifugal 15 min of 12000 rpm.Suct and reset and add 2 times of volume dehydrated alcohol-20 ℃ precipitation (sedimentation time 1.5 h).After precipitation finishes, centrifugal 15 min of 12000 rpm.With 70% washing with alcohol precipitation hypsokinesis, go, room temperature is dried.10 μ l TE(or aseptic ultrapure water for DNA that every duplicate samples is carried) dissolve, 20 ℃ save backup.
By following LAMP reaction system and reaction conditions, with designed primer, detect:
1. LAMP reaction system 25 μ l: comprise each 0.2 μ M of F3 and B3, FIP and BIP be 1.6 μ M respectively, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. in the final amplified production of LAMP reaction, add 1 μ l developer, described developer is SYBR green I, colour developing result is observed green fluorescence, or get 2 μ l amplified productions and detect with 2% agarose gel electrophoresis, there is the distinctive trapezoid belt of LAMP in result, can judge in described cucumber tissue or soil and have cucumber phytophthora; Otherwise there is not cucumber phytophthora in described cucumber tissue or soil.
Beneficial effect of the present invention: the inventive method is applicable to fast and reliable detection and the evaluation of cucumber phytophthora in incidence tissue or soil, has important practical value for the disease control that in agriculture production, cucumber phytophthora causes.The present invention compared with prior art, has following technical superiority and positively effect:
1, reliable results: the designed LAMP going out of the present invention detects primer, to coming from the cucumber phytophthora on the ground such as Fujian China, Guangdong, Jiangsu and having carried out testing authentication with soil sample, the plant tissue of cucumber phytophthora, so result reliability has sufficient assurance;
2, high specificity: LAMP primer of the present invention is for cucumber phytophthora
yptin gene order, 6 different zones are designed 4 Auele Specific Primers, and in 6 regions, any region is not mated all and can not be carried out nucleic acid amplification with primer, therefore specificity is high.
3, highly sensitive: LAMP can reach 10fg to the detection sensitivity of cucumber phytophthora on DNA level, than conventional PCR, detect high 1000 times.
4, practicality is good: the designed LAMP primer going out of the present invention, can be used for the highly sensitive rapid detection with tissue and the soil of cucumber phytophthora, therefore present method is practical, can meet the needs that the cucumber phytophthora with existing in hyphostroma and soil carried out to fast and reliable detection and evaluation;
5, easy and simple to handle quick: application the inventive method, tissue with cucumber phytophthora and soil are detected and can in 1 hour, be completed, and LAMP nucleic acid amplification is to carry out under isothermal condition, only need a water-bath, do not need complicated plant and instrument and expensive molecular agents, result naked eyes are directly visible.
Accompanying drawing explanation
Fig. 1 is the special LAMP detected result figure of Causal Organism of Cucumber Blight of the present invention.In Fig. 1, A represents agarose gel electrophoresis detected result, wherein: swimming lane M is DL 2000 DNA marker, the negative contrast of swimming lane 1, the positive contrast of swimming lane 2, swimming lane 3-8 is cucumber phytophthora, swimming lane 9-16 is other oomycetes and fungal bacterial strain; In Fig. 1, B represents the result that develops the color, wherein: the negative contrast of the 1st pipe, the positive contrast of the 2nd pipe, the 3rd – 8 pipes are cucumber phytophthora, the 9th – 16 pipes are other oomycetes and fungal bacterial strain.
Fig. 2 is the LAMP susceptibility detected result figure of Causal Organism of Cucumber Blight of the present invention.In Fig. 2, A represents agarose gel electrophoresis detected result, and wherein: swimming lane M is DL 2000 DNA marker, swimming lane 1 – 10 template concentrations are respectively 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg; In Fig. 2, B represents the result that develops the color, wherein: the 1st – 10 pipe template concentrations are respectively 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg.
Fig. 3 is the detected result figure of the present invention to disease plant and potato piece.In Fig. 3, A represents agarose gel electrophoresis detected result, wherein: the positive contrast of swimming lane 1, the negative contrast of swimming lane 2, swimming lane 3 is incidence tissue, and swimming lane 4 is morbidity soil, and swimming lane 5 is health tissues; Swimming lane M is DL 2000 DNA marker; In Fig. 3, B represents the result that develops the color, wherein: the positive contrast of the 1st pipe, the negative contrast of the 2nd pipe, the 3rd Guan Wei incidence tissue, the 4th pipe is morbidity soil, the 5th pipe is health tissues.
Embodiment
Technology contents of the present invention comprises that the LAMP of cucumber phytophthora detects primer, and LAMP primer and sequence thereof are respectively:
F3：5’-AAATTCGCACGATCGAGCT-3’
B3：5’-CCGTGACGTCGTACACCA-3’
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’
Result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis to utilize LAMP primer detection cucumber phytophthora to develop the color.
Main agents:
bstdNA polysaccharase large fragment is purchased from Britain NEB company; DNA marker is purchased from precious biotechnology Dalian company limited; All the other reagent are all purchased from Shanghai Sheng Gong Bioisystech Co., Ltd.Primer is synthetic by Shanghai Sheng Gong Bioisystech Co., Ltd.
the specific amplification of embodiment 1:LAMP primer pair cucumber phytophthora
1. the LAMP specific detection of cucumber phytophthora
1. LAMP reaction system 25 μ l: comprise each 0.2 μ M of F3 and B3, FIP and BIP be 1.6 μ M respectively, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. in the final amplified production of LAMP reaction, add 1 μ l developer, described developer is SYBR green I, and colour developing result is observed green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get 2 μ l amplified productions and detect with 2% agarose gel electrophoresis, if there is the distinctive trapezoid belt of LAMP, be judged as the positive, do not occur that amplified band is judged as feminine gender.
2. detected result
The specificity detecting: results can be observed green fluorescence or agarose gel electrophoresis occurs the distinctive trapezoid belt of LAMP except the 36 strain cucumber phytophthoras from provinces such as China Fujian, Guangdong, Jiangsu develop the color, detected 11 kinds of other oomycetes and 22 kinds of fungal bacterial strains colour developing result is orange or amplified band (partial results is shown in Fig. 1) does not appear in agarose gel electrophoresis, illustrated that this primer has very strong specificity.
the susceptibility of embodiment 2:LAMP primer pair cucumber phytophthora detects
1. the LAMP susceptibility of cucumber phytophthora detects
Adopt 10 times of concentration series dilution methods that the cucumber phytophthora DNA of extraction is diluted to 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg is totally 10 different concns gradients.
1. LAMP reaction system 25 μ l: comprise each 0.2 μ M of F3 and B3, FIP and BIP be 1.6 μ M respectively, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. in the final amplified production of LAMP reaction, add 1 μ l developer, described developer is SYBR green I, and colour developing result is observed green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get 2 μ l amplified productions and detect with 2% agarose gel electrophoresis, if there is the distinctive trapezoid belt of LAMP, be judged as the positive, do not occur that amplified band is judged as feminine gender.
2. detected result: cucumber phytophthora LAMP susceptibility detects, colour developing result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, detection sensitivity can reach 10fg(and see Fig. 2).
embodiment 3: the detection of cucumber phytophthora in incidence tissue or soil.
1. sample collecting: plant tissue sample picks up from cucumber production base, Fuzhou City, Fujian; Pedotheque picks up from cucumber production base, Longhai City, Fujian Province.
2.DNA extracts and detects
Morbidity plant tissue adopts the quick cracking process of NaOH to extract cucumber phytophthora DNA, and morbidity soil adopts soil DNA extraction method to extract the DNA of cucumber phytophthora.
Carry out as follows LAMP detection:
1. LAMP reaction system 25 μ l: comprise each 0.2 μ M of F3 and B3, FIP and BIP be 1.6 μ M respectively, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 25ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 63 ℃ of incubation 60 min, 80 ℃ of insulation 10min.
2. in the final amplified production of LAMP reaction, add 1 μ l developer, described developer is SYBR green I, and colour developing result is observed green fluorescence and is judged as the positive, the orange feminine gender that is judged as.Or get 2 μ l amplified productions and detect with 2% agarose gel electrophoresis, if there is the distinctive trapezoid belt of LAMP, be judged as the positive, do not occur that amplified band is judged as feminine gender.
3. detected result
As shown in Figure 3, in incidence tissue or soil, infect cucumber phytophthora, colour developing result can be observed green fluorescence or the distinctive trapezoid belt of LAMP appears in agarose gel electrophoresis, and health tissues colour developing result observes fluorescent orange or the distinctive trapezoid belt of LAMP does not appear in agarose gel electrophoresis.
<110> Inst. of Plant Protection, fujian Academy of Agricultural Science
<120> cucumber phytophthora LAMP primer and method for quick thereof
<160> 4
<170> PatentIn?version?3.5
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
aaattcgcac?gatcgagct 19
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
ccgtgacgtc?gtacacca?18
<210> 3
<211> 41
<212> DNA
<213> artificial sequence
<400> 3
gcgctaagtc?gcgaatgtac?cggacggcaa?gaccatcaag?c?41
<210> 4
<211> 41
<212> DNA
<213> artificial sequence
<400> 4
ctattgtagt?gggacacggc?cgcgataata?ccgtgggcac?c?41
Claims (3)
1. a LAMP primer for cucumber phytophthora (Phytophthora melonis), is characterized in that: described LAMP primer is as follows:
F3：5’-AAATTCGCACGATCGAGCT-3’?；
B3：5’-CCGTGACGTCGTACACCA-3’；
FIP：5’-GCGCTAAGTCGCGAATGTACCG-GACGGCAAGACCATCAAGC-3’；
BIP：5’-CTATTGTAGTGGGACACGGCCG-CGATAATACCGTGGGCACC-3’。
2. the LAMP detection method of a cucumber phytophthora, it is characterized in that: utilize the LAMP primer described in claim 1 to carry out LAMP reaction, each 0.2-0.25 μ M of F3 and B3 in 25 μ l reaction systems, each 1.5-1.7 μ M of FIP and BIP, 20mM Tris-HCl, 10mM (NH
4)
2sO
4, 10mM KCl, 8mM MgSO
4, 0.1% Tween-20,0.8M Betaine, 1.4 mM dNTPs, 8U
bstdNA polysaccharase large fragment, 10-50ng DNA profiling, insufficient section is supplied with aseptic double-distilled water; LAMP reaction conditions is at 60-65 ℃ of incubation 30-90 min, 80 ℃ of insulation 5-10min.
3. cucumber phytophthora LAMP detection method according to claim 2, it is characterized in that: in the amplified production of LAMP reaction, add 1 μ l developer, described developer is SYBR green I, and colour developing result is observed green fluorescence and is judged as the positive, the orange feminine gender that is judged as; Or get 2 μ l amplified productions and detect with 2% agarose gel electrophoresis, as occurred, trapezoid-shaped strips is judged as the positive, does not occur being judged as feminine gender.
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| CN104962639B (en) * | 2015-07-09 | 2019-01-15 | 天津市植物保护研究所 | A kind of the LAMP primer group and detection method of the how main stick spore bacterium of quick detection |
| CN105039331B (en) * | 2015-08-11 | 2018-01-26 | 华南农业大学 | A kind of peronophythora litchi LAMP primer and its quick determination method and application |
| CN106987653B (en) * | 2017-06-09 | 2021-04-09 | 福建省农业科学院植物保护研究所 | LAMP (loop-mediated isothermal amplification) detection primer for potato late blight bacteria and visual detection method thereof |
| CN108103140B (en) * | 2017-11-09 | 2021-04-06 | 广东省农业科学院蔬菜研究所 | Method for rapidly identifying resistance of cucumber to epidemic disease |
| CN109486985A (en) * | 2018-08-07 | 2019-03-19 | 天津出入境检验检疫局动植物与食品检测中心 | The LAMP primer pair and its amplification method and purposes of detection phytophthora hibernalis bacterium |
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